Project Summary While a number of studies have defined how inflammation arises and is maintained, much less is known about how it is resolved. We have found that colon epithelium is key to either exacerbating or suppressing inflammation. It is well-established that microbiota and immune cells signal to epithelial cells to increase production of chemokines and pro-inflammatory cytokines that can accelerate inflammation, but we have found that TGFb signaling via SMAD4 within the epithelium suppresses a pro-inflammatory transcriptional network. After conditional Smad4 gene deletion in the adult murine colonic epithelium, we found striking evidence for increased inflammatory signaling within the epithelial compartment concomitant with an increase in inflammatory infiltrate. Mechanistically, TGFb1 and/or BMP2 inhibit transcriptional induction of multiple inflammatory genes by TNF, IL- 1b, or LPS treatment in mouse and human cultured colon epithelial cells. Furthermore, >75% of mice with adult- onset deletion of the Smad4 gene in intestinal epithelium developed invasive mucinous adenocarcinomas of the distal colon within 3 months after dextran sodium sulfate (DSS)-induced colitis, while no tumors were found without colitis or in the SMAD4+ controls. In humans, we found a much higher incidence of SMAD4 loss in colitis- associated carcinoma (CAC) than in sporadic colorectal cancer. Based on our observations, we hypothesize that TGFb signaling via SMAD4 functions as a tumor suppressor, in part, through the inhibition of pro-inflammatory cytokine responses in intestinal epithelial cells. The major goal of this research is to determine the mechanisms by which TGFb superfamily signaling regulates colonic inflammatory responses and how this regulation is linked to tumor suppression. We will examine our hypothesis through the following specific aims: Aim 1. Determine the mechanism by which SMAD4-mediated signaling modulates cytokine signaling pathways in colon epithelial cells. Working hypothesis: SMAD4-mediated signaling inhibits transcriptional responses to inflammatory stimuli in colonic epithelial cells via specific inhibitory complexes on cytokine-induced genes. We will determine which genes are directly regulated by SMAD4 and how this regulation alters responses to pro-inflammatory cues in cultured cells and in vivo through a combination of RNA-seq, ATAC-seq and ChIP-seq experiments. Aim 2. Determine how dysregulated cytokine signaling regulates tumorigenesis in Smad4 null colonic epithelium. Working hypothesis: SMAD4-mediated suppression of epithelial inflammatory response genes is required to prevent tumorigenesis during colitis. We propose to examine in vivo how SMAD4 regulates specific inflammation- associated signaling pathways and gene expression and how those pathways contribute to suppression of CAC. We will identify these regulatory pathways using single cell analytical approaches to understand the cell-specific roles of SMAD4 within colon epithelium and will evaluate the role of SMAD4 in suppressing epithelial inflammatory genes in human CRC, particularly in CAC. Our work will yield better understanding of inflammatory regulation in colonic epithelium and is likely to identify novel regulatory targets for interventional strategies.